The present invention relates to organic polymer particles and a process for producing the same, magnetic particles for diagnostics, and carboxyl group-containing particles and a process for producing the same.
The invention further relates to probe-bound particles comprising the organic polymer particles with a probe bound therewith and a process for producing the same.
Organic polymer particles and magnetic particles are used as a reaction solid phase of a diagnostic agent using an antigen-antibody reaction in order to detect substances to be examined such as infections, cancer markers, hormones, and the like. In such a diagnostic agent, a probe (primary probe) for inspecting an antibody or an antigen is immobilized on particles. A substance to be inspected in a sample reacts with a second inspection probe after having been caught by the particles via the primary probe. The second inspection probe (secondary probe) is labeled with a fluorescent substance or an enzyme, whereby the target substance is detected by fluorescence or by an enzyme reaction.
In recent years, due to a demand for an increase in the inspection sensitivity for early detection of diseases, an increase in sensitivity of a diagnostic agent has been an important subject. In order to increase sensitivity of diagnostic agents using magnetic particles, a method of using enzyme coloring as a detecting means is being replaced by a method of using fluorescence or chemiluminescence, both of which ensure higher sensitivity.
Development of these detection techniques are said to have reached a level in which a one molecule-detection target can be theoretically detected. In practice, however, sensitivity is still insufficient. One reason for the insufficient sensitivity is non-specific adsorption of secondary probes and impurities onto the surface of particles. For example, even if a technique that can theoretically detect a one molecule-detection target is used, detection of the one molecule is impossible if several molecules of a secondary probe are non-specifically adsorbed onto the surface of the particles. For this reason, a technique for controlling non-specific adsorption onto the particle surface of substance used for inspection is strongly demanded.
A blocking method has been used for controlling such non-specific adsorption. In the blocking method, after immobilizing a primary probe on the particles, the particle surface is covered with a blocking agent such as albumin or skim milk with minimal adsorptivity of a secondary probe, impurities, and the like. However, some blocking agents may not exhibit a sufficient effect of covering. Other blocking agents, which are biological substances, exhibit only poor quality stability. In some cases, a sufficient effect of controlling non-specific adsorption cannot be obtained even if the particle surface is adequately covered with a blocking agent, because the blocking agent loses its effect over time due to denaturing and the like.
In order to solve the problem of non-specific adsorption, a method of introducing a hydrophilic polymer onto the surface of a substrate for immunoassay represented by a 96-well plate has been proposed (JP-A-1-174057, JP-A-2000-304749, and JP-A-2001-272406). However, because the area available for immobilizing a primary probe is limited and the reaction of a primary probe with the target substance to be detected is a solid-liquid reaction, such an immunoassay substrate utilizing a plane has problems of poor efficiency of an antigen-antibody reaction, a long period of time required for inspection, and the like.
Furthermore, as countermeasures for decreasing non-specific adsorption, microspheres made from organic polymer particles of a styrene-glycidyl methacrylate copolymer and the like and a physiological active substance bonded to the organic polymer particles via a spacer (JP-A-10-195099, JP-A-2000-300283, WO 04/025297), organic polymer particles with a hydrophilic spacer introduced on the particle surface (JP-A-2004-331953, WO 04/040305), and the like have been proposed. These organic polymer particles, however, exhibited neither a sufficient effect of reducing non-specific adsorption nor sufficient immunoassay sensitivity.
The present inventors have proposed magnetic particles for immunoassay exhibiting almost no non-specific adsorption, the particles having hydrophilic monomers such as hydroxyalkyl (meth)acrylate, alkoxyalkyl (meth)acrylate, polyoxyalkylene (C2-C4) group-containing (meth)acrylate, epoxy group-containing (meth)acrylate, phosphorylcholine-analogous group-containing monomers, and the like copolymerized on the surface (JP-A-2005-69926). However, development of particles for immunoassay exhibiting higher sensitivity have been desired. If a large amount of hydrophilic functional groups are introduced in order to decrease non-specific adsorption, the viscosity of aggregates of magnetic particles (hereinafter referred to as “pellets”) after magnetic separation unduly decreases and magnetic particles may migrate into the supernatant solution which is removed after the magnetic separation.
In addition, when a probe (a primary probe) for detecting proteins, such as an antibody or an antigen, and nucleic acid is bound to the particle surface, the particles may aggregate during the reaction.